INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Protein-tyrosine phosphorylation plays a vital role in many cellular processes including growth and differentiation (18, 54). Cellular levels of tyrosine phosphorylation are maintained by a balance between protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) activity (18). At present, more than 75 PTP family members have been identified, and it has been suggested that the human genome could encode more than a hundred PTPs (54). The PTP superfamily is subdivided into three subfamilies: the dual-specificity PTPs, the intracellular PTPs, and the receptor-like PTPs (RPTPs) (49). Most RPTPs have tandem catalytic domains, with the majority of catalytic activity residing in the membrane-proximal catalytic domain (D1). While it is well established that ligand binding to receptor PTKs results in dimerization, transautophosphorylation, and kinase activation (16), how the activity of RPTPs is regulated remains poorly understood. Only a handful of RPTPs have been found to bind to other proteins via their extracellular domains (ECDs), and until recently none of these interacting proteins had been found to modulate the activity of the cognate RPTP (1, 34, 38, 40, 41, 62). However, the discovery that the secreted factor pleiotrophin interacts with and inhibits the activity of RPTP (also called RPTB) in vitro and in vivo (33) indicates that regulatory ligands for RPTPs do exist.

Based upon emerging structural and functional evidence, it has been proposed that, whereas dimerization activates receptor PTKs, dimerization may inhibit RPTPs (61). In two independent crystal forms the membrane-proximal catalytic domain (D1) of murine RPTP exists as a symmetric dimer, in which a helix-turn-helix wedge-shaped element on each monomer inserts into the active site of the dyad-related monomer, resulting in mutual active-site occlusion (3). In principle, RPTP dimers of this sort would lack catalytic activity, and a fraction of RPTP elutes from gel filtration columns with a size larger than expected for a monomer, suggesting that RPTP may indeed have the ability to dimerize or oligomerize (6). Recently, we showed that RPTP containing a Pro137Cys mutation in the ECD dimerizes constitutively via a disulfide bond and has greatly reduced biological activity in vivo, providing the evidence that dimerization can indeed inhibit the biological activity of a full-length RPTP (20). Consistent with this, EGF-induced dimerization functionally inhibits the biological activity of an EGF receptor-CD45 chimera expressed in a T-cell line in a wedge-dependent fashion (11, 31). Moreover, RPTP D2 inhibits RPTP-D1 activity in vitro (58).

Although artificial dimerization can inhibit the biological activity of some RPTPs, whether RPTPs dimerize physiologically and whether this results in functional inhibition is largely unknown. CD45, which is required for T- and B-cell receptor signaling, has been chemically cross-linked in lysates and to a lesser extent in intact cells (53), and recombinant CD45 cytoplasmic domain dimerizes in solution, emphasizing the dimerization potential of CD45 (13). Moreover, mutation of the wedge motif in CD45 in the mouse germ line leads to an immunoproliferative syndrome in vivo (R. Majeti and A. Weiss, personal communication), implying that wedge-mediated CD45 dimerization suppresses CD45 biological activity. However, even though a conserved wedge motif is present upstream of D1 in most RPTPs, not all RPTPs may form dimers in a manner similar to RPTP and CD45. For instance, D1 of RPTPµ does not exist as a wedge-mediated dimer in the crystal structure (17), nor is the cytoplasmic region (D1+D2) of LAR present as a dimer in the crystal structure (37). Therefore, it is important to determine whether RPTPs form dimers in the cell and to understand the structural basis for dimers, if they exist.

In this study, we have used RPTP as a model system to investigate both the efficiency and the structural determinants of homodimerization in vivo. RPTP contains a rather short 123-amino-acid N-terminal ECD, a single transmembrane domain (TMD), two intracellular PTP domains D1 and D2 (see Fig. 1) (21, 24, 32, 45). Unlike most RPTPs, in which only D1 is catalytically active, both PTP domains in RPTP are active, although D1 possesses substantially greater catalytic activity than does D2 (5, 29, 30, 59). RPTP is widely expressed in mammalian tissues (41) and has been implicated in a variety of signaling pathways (2, 7, 8, 10, 19, 27, 36, 51, 56, 63, 65). For example, RPTP has been shown to play a role in both cellular differentiation and cellular transformation by directly dephosphorylating phosphorylated Tyr527 in c-Src, leading to enhanced c-Src catalytic activity (8, 65). Recently, it was shown that RPTP null cells (RPTP^/ cells) derived from RPTP knockout mice have greatly reduced c-Src PTK activity and are defective in cell adhesion and spreading, all of which are restored upon ectopic expression of RPTP (42, 50). Moreover, the binding of the c-Src SH2 domain to the C-terminal P.Tyr789 in RPTP results in displacement of P.Tyr527 from the SH2 domain, thus allowing RPTP to dephosphorylate P.Tyr527 and thereby specifically activate c-Src (64). RPTP has been found to be overexpressed in late-stage colon carcinomas (52), where c-Src is commonly found to be activated. RPTP localization to focal adhesions requires Tyr789 at the C terminus (26), and p130Cas, which is localized to focal adhesions, has recently been shown to interact with and be a substrate for RPTP (4). No ligand has been found for RPTP, but RPTP interacts with the GPI-linked protein contactin in neuronal cells to form a complex that may be linked to the intracellular Src family PTK Fyn (62). Although no regulatory ligand is known, RPTP in vitro activity is enhanced by tetradecanoyl phorbol acetate treatment of cells, which results in protein kinase C-mediated phosphorylation of Ser180 and Ser204 (9, 55).

Based on surface cross-linking studies, we provide the first evidence that RPTP homodimerizes efficiently on the cell surface via multiple domains, suggesting that dimerization-mediated negative regulation of RPTP biological activity is likely to be physiologically relevant.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression vectors, site-directed mutagenesis, and antisera. The expression vector pSG5 was previously described (14). All constructs used in this study were subcloned into pSG5. Construct ut.FL corresponds to the untagged wild-type full-length murine RPTP. Construct FL corresponds to a full-length murine RPTP with a hemagglutinin (HA) epitope inserted between amino acids 19 and 20 (Fig. 1) (7, 8). Construct FL.137C contains a Pro137Cys single-amino-acid substitution (Fig. 1) (20). Construct Myr.Cyto corresponds to a myristoylated form of the RPTP cytoplasmic domain, containing residues 163 to 794 of RPTP and an N-terminal myristoylation signal (residues 1 to 11 of mouse c-Src) (Fig. 1). To prepare this construct, a HindIII/BsrGI fragment (encoding residues 1 to 162 of RPTP) of the RPTP cDNA in the pSG.HA.RPTP expression vector was replaced with a double-stranded oligonucleotide composed of a sense strand (5'-agctt gccgac ATG GGG AGT AGC AAG AGC AAG CCT AAG GAC CCC ct-3'; an HindIII site-compatible end is underlined; the initiation codon is italicized; the sequence coding for c-Src amino acid residues 1 to 11 is capitalized) and the antisense strand (5'-gta cag GGG GTC CTT AGG CTT GCT CTT GCT ACT CCC CAT gtc ggc a-3'; the BsrGI-compatible end is underlined; the antisense sequence of residues 1 to 11 of c-Src is capitalized).

Cell culture and transient transfection. HEK293 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum at 37°C and 10% CO₂. Transient transfection of 293 cells was done using the calcium phosphate precipitation method. Briefly, cells were seeded onto 50-mm tissue culture dishes with or without poly-L-lysine coating 24 h prior to transfection at the dilution of 1 confluent 100-mm dish to 20 50-mm dishes. Poly-L-lysine does not affect cross-linking of RPTP (Fig. 2C) but prevents cells from detaching during the many solution changes in the cross-linking procedure. At the start of transfection (0 h), 4 ml of fresh medium containing 25 µM chloroquine was added to each of the dishes. Plasmid DNA was mixed with 500 µl of 250 mM CaCl₂, to which 500 µl 2× HBS (50 mM HEPES; 10 mM KCl; 280 mM NaCl; 1.5 mM Na₂HPO₄; 12 mM dextrose, pH 7.05) was subsequently added. The mix was immediately added to the medium, and cells were then incubated at 37°C and 5% CO₂. At 10 h, the cells were washed twice with phosphate-buffered saline (PBS) and incubated in fresh medium at 37°C and 10% CO₂. Chemical cross-linking or cell surface biotinylation were performed at approximately 72 h.

Cell surface chemical cross-linking. The entire procedure was performed on intact cells at 4°C. Transiently transfected 293 cells in 50-mm dishes were washed three times with PBS and incubated with freshly prepared cross-linker solution [bis(sulfosuccinimidyl)suberate (BS³; Pierce) at 3 mg/ml in PBS (pH 7.1) without Ca²⁺ and Mg²⁺] for ~60 min and then washed three times with PBS and incubated in PBS solution containing 50 mM Tris-HCl (pH 7.5) for 20 min to quench residual BS³. The cells were then lysed in radioimmunoprecipitation assay (RIPA) buffer (58) for 30 min. The lysates were cleared by centrifugation, and the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation, the proteins on the gels were transferred to Immobilon (Millipore, Bedford, Mass.) and subjected to immunoblotting to detect the RPTP proteins. To visualize the dimeric or monomeric RPTP proteins, membranes were probed with affinity-purified polyclonal antisera 5478 or MAb 12CA5 and detected by enhanced chemiluminescence (ECL) as previously described (7). To quantitate the monomers and dimers, membranes were probed with MAb 12CA5 as above, washed three times with TBST (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween 20), blocked with TBST containing 5% milk for 30 min, washed once with TBST, and blocked with TBST containing 1% bovine serum albumin (BSA) for 20 min. ¹²⁵I-labeled sheep anti-mouse immunoglobulin G (IgG) F(ab')₂ fragment (NEN Life Science Products, Inc.) was then added to the blocking solution at a final concentration of 0.5 µCi/ml, and the incubation was continued for another 1.5 h. The membranes were then washed three times with TBST, dried, and analyzed using a PhosphorImager (Molecular Dynamics).

Cell surface biotinylation. To biotinylate surface proteins, transiently transfected HEK293 cells in 50-mm dishes were washed three times with PBS, incubated for 20 min with freshly prepared biotinylation buffer (50 mM sodium phosphate; 110 mM NaCl; 0.1% NaN₃, pH 8.5) containing EZ-Link-Sulfo-NHS-LC-Biotin (Pierce) at 0.4 mg/ml, and washed three times with PBS containing 0.1% NaN₃. Cells were then lysed in RIPA buffer. The level of biotinylated RPTP was determined by two different methods. In the first method, total RPTP protein was isolated by immunoprecipitation from whole-cell lysates, and the biotinylated RPTP was then detected by immunoblotting using ¹²⁵I-labeled streptavidin (Amersham). Briefly, the lysates were incubated with MAb 12CA5 (approximately 0.5 to 1.0 µg of IgG per sample) for 1 h with gentle rotation. Protein A-Sepharose beads (25 µl of 50% bead suspension solution per sample) were subsequently added, and the incubation was continued for another 1.5 h. Beads were then washed three times with RIPA buffer and boiled in Laemmli sample buffer. The immunoprecipitates were separated by SDS-PAGE and, after separation, the proteins on the gel were transferred to Immobilon. The membranes were blocked with TBST containing 5% milk at 4°C overnight for 30 min, washed once with TBST, and then blocked with TBST containing 1% BSA for 20 min. ¹²⁵I-labeled streptavidin was then added to the blocking solution. The incubation was continued for another 1.5 h. The membranes were then washed three times with TBST and analyzed by PhosphorImager analysis. In the second method, biotinylated proteins were isolated using streptavidin-agarose beads (Sigma) by the procedures described above. After SDS-PAGE, the level of biotinylated RPTP was then detected and quantified by immunoblotting using MAb 12CA5 and secondary antiserum ¹²⁵I-labeled sheep anti-mouse IgG F(ab')₂ fragment as described above. We have optimized the biotinylation experiments using different incubation times and biotin concentrations. The conditions described above represent those under which biotinylation efficiency is maximized.

Inhibition of glycosylation in vivo and deglycosylation in vitro. To inhibit N-linked glycosylation, transfected HEK293 cells were washed three times with PBS 12 h after the initiation of transfection and then incubated in fresh medium containing either control solvent (dimethyl sulfoxide) or tunicamycin (Sigma) at the desired concentration. After incubation for another 12 h, the cells were cross-linked and/or lysed in lysis buffer (50 mM HEPES, pH 7.0; 150 mM NaCl; 1.5 mM MgCl₂; 1 mM EGTA; 10% [vol/vol] glycerol; 1% Triton X-100; 1.0 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). For in vitro deglycosylation, 5 to 30 µl of the lysates were incubated with 1 to 4 µU of either N-glycosidase F (Sigma) to remove N-linked glycosyl groups or endo--N-acetylgalactosaminidase (Sigma) to remove O-linked glycosyl groups.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

RPTP oligomerizes on the cell surface with high efficiency. We showed previously that RPTP D1 exists as a dimer in two independent crystal forms (3). To determine whether RPTP dimerizes on the cell surface, BS³-mediated chemical cross-linking was performed on intact 293 human embryonic kidney cells (293 cells) transiently transfected with an N-terminally HA-tagged full-length RPTP expression vector and plated on poly-L-lysine (FL; Fig. 1A). BS³, which reacts with free NH₂ groups in proteins, is not membrane permeant due to its charged nature and therefore only cross-links surface-expressed proteins. Using MAb 12CA5, which specifically recognizes the HA tag, immunoblotting analysis of whole-cell lysate of mock-cross-linked cells showed that FL RPTP migrated as an ~130-kDa band (Fig. 1B, lane 1), representing fully glycosylated FL protein. Immunoblotting analysis of whole-cell lysate of BS³-cross-linked cells revealed an additional band of ~230 kDa (Fig. 1B, lanes 2), indicating that RPTP oligomerizes on the cell surface. The apparent size of the ~230-kDa band suggests that it contains RPTP homodimers.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   RPTP homodimerizes on the cell surface. (A) A schematic of RPTP constructs used in this figure. Amino acids are numbered, and boundaries of the various structural domains, including the TMD, D1, and D2, are indicated according to the original (untagged) polypeptide (45). The boundary of the wedge region is indicated according to the D1 crystal structure (3). The construct FL contains an HA tag which was inserted between amino acids 19 and 20 and is exposed by signal peptide cleavage. The construct ut.FL corresponds to the full-length untagged RPTP. The construct Myr.Cyto contains a myristoylation signal for membrane attachment. For details of vector construction, refer to Materials and Methods. Mock cross-linking and cross-linking on intact 293 cells transiently expressing FL (B and E), ut.FL (C), and Myr.Cyto (D) is shown. (E) Cells were cultured on plates without or with poly-L-lysine coating. Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates (WCL) using ECL detection (B and E) or polyclonal antiserum 5478 (C and D). BS3: , mock cross-linking without BS3; +, cross-linking with BS3. Tun, transfected 293 cells were exposed to tunicamycin at 200 ng/ml; F, lysate was deglycosylated with N-glycosidase F in vitro; , lysate was deglycosylated with endo--N-acetylgalactosaminidase in vitro. M, monomers; D, dimers. The positions of RPTP monomer (M) and dimer (D) are indicated at the right side of the figure. The positions of molecular-weight markers (in kilodaltons) are indicated on the left side of the figure. Similar labels are used throughout the study.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   RPTP appears to exist on the cell surface predominantly as homodimers. (A) 293 cells transiently expressing FL were surface biotinylated or not biotinylated. Cells were lysed, and biotinylated proteins were precipitated with streptavidin beads and separated by SDS-PAGE, and the biotinylated FL was detected by immunoblotting using MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2. The blot was quantified using a PhosphorImager as described in Materials and Methods. Top and bottom panels are the immunoblot and quantification, respectively. The loading for each of the lanes was standardized using an equivalent amount of whole-cell lysate. Lane 7 is a longer exposure of lane 6. + or  biotin, labeled or not labeled with biotin; WCL, total whole-cell lysate; SN, whole-cell lysate supernatant after streptavidin bead precipitation; P, streptavidin precipitate. (B) BS3 cross-linking was performed on 293 cells transiently transfected with either FL or FL.137C. Whole-cell lysates of cross-linked cells were separated by SDS-PAGE and probed using MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2 and then quantified using a PhosphorImager as described in Materials and Methods. Shown are the results of an immunoblotting analysis. The right panel shows quantitation of the gel in left panel. n, number of replicates. Note that FL and FL.137C are similarly localized to the cell surface (20).

The wedge structure is important but not essential for RPTP oligomerization. RPTP D1 dimers in crystals are stabilized by interactions between the active site of one monomer and the wedge of the dyad axis-related monomer. The wedge structure itself is stabilized by residues P210 and P211 at the base, and several residues at the tip, including E234, participate in protein-protein interactions (3). We showed that FL.137C dimerizes in vivo and has reduced biological activity. Furthermore, the biological activity of FL.137C can be restored by the P210L.P211L double mutation but not by other mutations, including E234A in the wedge, probably because the P210L.P211L mutation causes a more significant structural effect (20). These results suggest, both structurally and functionally, that the wedge is important for RPTP oligomerization. Accordingly, the cross-linking efficiency of FL was compared to that of several wedge mutants, including FL.P210L.P211L, FL.E234A, and 224-235, a deletion that is predicted to eliminate the entire wedge structure (Fig. 3A). When the wild type (FL) and the wedge mutants were expressed to similar levels on the cell surface as determined by biotinylation (Fig. 3B, top panel), dimeric forms of the proteins were readily detectable for FL and FL.E234A but not for FL.P210L.P211L and 224-235 (Fig. 3B, bottom panel, lanes 1 and 3 versus lanes 2 and 4). Quantitative analysis demonstrated that both the P210L.P211L double mutation and the wedge deletion reduced oligomerization efficiency of RPTP by approximately 80% on the cell surface (Fig. 3C). The fact that the 224-235 and FL.P210L.P211L proteins have reduced dimerization potential indicates that the wedge structure is important for RPTP homodimerization. The result that the 224-235 and FL.P210L.P211L proteins dimerized with similarly low efficiency confirmed our previous speculation that the P210L.P211L double mutation likely disrupts the wedge structure. Finally, the finding that 224-235 mutant still dimerized suggests that there are other oligomerization domain(s) in addition to the wedge in RPTP. The fact that the wedge mutations affect RPTP cross-linking in a fashion stereochemically consistent with the crystallographic data confirmed that BS³-mediated cross-linking of RPTP is specific.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Mutations in the wedge diminish but do not abolish RPTP oligomerization. (A) A schematic of RPTP wedge mutant constructs, including point mutants FL.P210L.P211L and FL.E234A and deletion mutant 224-235. (B) For the top panel, transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTP proteins, which were then subjected to SDS-PAGE and probed with 125I-labeled streptavidin to determine the levels of surface-expressed RPTP protein. For the bottom panel, transiently transfected 293 cells were cross-linked with BS3. Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2 to determine the levels of RPTP dimers. The bands representing FL.P210L.P211L dimers and 224-235 dimers are faint but detectable by PhosphorImager analysis. Biotinylation and cross-linking were done on parallel dishes from the same transfection. All the constructs were expressed to a similar level on the cell surface. (C) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTP dimers over surface-expressed RPTP, which were determined from the bottom and top portions of panel B, respectively, using a PhosphorImager. S, surface-expressed RPTP (monomer).

The C-terminal catalytic domain D2 is important but not essential for RPTP oligomerization. To investigate the possibility that RPTP has oligomerization domains in addition to the wedge, we tested the oligomerization potential of an RPTP deletion mutant lacking the C-terminal D2 (D2) (Fig. 4A). Immunoblotting analysis of 293 cells transiently transfected with the D2 expression vector showed that MAb 12CA5 specifically recognized a band of ~100 kDa (Fig. 4B, lane 1), a finding consistent with the predicted size of a fully glycosylated D2 protein. After cross-linking, a new band of ~160 kDa was detected (Fig. 4B, lane 2), indicating that D2 oligomerizes. To assess the dimerization potential of D2, we compared the dimerization efficiency of the D2 and wild-type FL proteins. When the two proteins were expressed on the cell surface to similar levels as determined by surface biotinylation (Fig. 4C, lanes 1 and 2 versus lanes 3 and 4), FL dimers but no D2 dimers were apparent (Fig. 4D, lanes 1 and 2 versus lanes 3 and 4), indicating that D2 has reduced cross-linking efficiency compared to FL. Taken together, these results suggest that D2 participates but is not essential for RPTP homodimerization.

View larger version (38K): [in this window] [in a new window]   FIG. 4.   Deletion of D2 diminishes but does not abolish RPTP oligomerization. (A) A schematic of the D2 deletion mutant construct. (B) 293 cells transiently expressing D2 protein were cross-linked or not cross-linked with BS3. Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTP proteins, which were then subjected to SDS-PAGE and probed with 125I-labeled streptavidin to determine the levels of surface-expressed RPTP protein. (D) Transiently transfected 293 cells were cross-linked with BS3. Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2 to determine the levels of RPTP dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images obtained via PhosphorImager analysis. S/M, surface-expressed monomeric proteins.

The TMD and the ECD also participate in RPTP homodimerization. Our data indicate that both D1 and D2 participate in RPTP homodimerization but that neither of them is essential. To determine whether the entire cytoplasmic domain is essential for RPTP homodimerization and whether the TMD and the ECD also participate in RPTP homodimerization, we made a construct, Cyto, which lacks the entire cytoplasmic domain but contains the ECD and the TMD (Fig. 5A). Immunoblotting analysis of 293 cells transiently transfected with the Cyto expression vector showed that MAb 12CA5 specifically recognized an ~80-kDa band (Fig. 5B, lane 1), most likely representing fully glycosylated Cyto protein. The expected molecular size of fully glycosylated Cyto is ~65 kDa (~40 kDa contributed by glycosylation) instead of the observed 80 kDa. The SDS gel mobility of a protein in which carbohydrate contributes most of the mass is hard to predict, but it seems likely that the protein will run significantly slower than expected because the charge/mass ratio of the carbohydrate to which SDS does not bind is expected to be lower than that of SDS-saturated protein. After cross-linking of Cyto-expressing cells, a new band of ~140 kDa was also detected (Fig. 4C, lane 2), indicating that Cyto oligomerized. Tunicamycin treatment in vivo to inhibit N-linked glycosylation caused a parallel reduction in the apparent size of the 80- and 140-kDa protein species (Fig. 4C, lane 3 versus lane 2), confirming that the ~140-kDa band is a Cyto oligomer (we show later that the Cyto oligomer is a homodimer [see Fig. 6 and associated text]).

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Cyto homodimerizes on the cell surface with high efficiency. (A) A schematic of the construct Cyto lacking the entire cytoplasmic domain. (B) 293 cells transiently transfected with FL or Cyto were treated or not treated with tunicamycin at 200 ng/ml and subsequently cross-linked with BS3. Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were precipitated with streptavidin beads to isolate the total RPTP proteins, which were then subjected to SDS-PAGE and probed with 125I-labeled streptavidin to determine the levels of surface-expressed RPTP proteins. (D) Transiently transfected 293 cells were cross-linked with BS3. Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2 to determine the levels of RPTP dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images from PhosphorImager analysis. S/M, surface-expressed monomeric proteins. (E) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTP dimers over surface-expressed RPTP, which were determined from panels C and D, respectively, using a PhosphorImager. n.s., nonspecific band.

View larger version (46K): [in this window] [in a new window]   FIG. 6.   RPTP oligomers are homodimers. Mock cross-linking and cross-linking on 293 cells transiently transfected was done with the construct FL alone (lane 1), with Cyto alone (lanes 2 and 3), or with both FL and Cyto simultaneously (lanes 4 and 5). Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. Note that a band corresponding to either partially glycosylated or degraded Cyto is present in the transfections of Cyto alone (lanes 2 and 3) but is virtually undetectable in the cotransfections (lanes 4 and 5). (FL+Cyto)H.D. is the cross-linked FL-Cyto heterodimer.

RPTP oligomers are homodimers. We concluded that the oligomers detected in the previous experiments are RPTP homodimers. However, the apparent sizes of most of the various oligomers judged by their migration in SDS-polyacrylamide gels are somewhat less than twice the sizes of the corresponding monomers (FL = ~130 kDa, FL oligomers = ~230 kDa; Cyto = ~80 kDa, Cyto oligomer = ~140 kDa). To rule out the formal possibility that the oligomers are RPTP heterodimers or hetero-oligomers with other proteins, the potential of FL and Cyto to heterodimerize was determined by a cross-linking experiment with 293 cells that had been cotransfected with both expression vectors. Immunoblotting analysis using MAb 12CA5 detected a novel band of ~200 kDa migrating between FL oligomers and fg.Cyto oligomers (Fig. 6, lane 5 versus lanes 1 and 3), suggesting that it was a FL-Cyto heterodimer. The results show that RPTP homodimerizes and that the oligomers observed in Fig. 1 to 5 are RPTP homodimers. We explain the fact that the dimers migrate faster than expected as most likely being due to the nonlinear backbone structures of the cross-linked molecules.

The RPTP ECD dimerizes weakly and is not essential for oligomerization. To determine whether the ECD participates in RPTP homodimerization, we determined the dimerization potential of ECD.GPI, a protein corresponding to RPTP ECD with a C-terminally fused GPI linkage signal sequence (Fig. 7A). The GPI membrane anchor for the ECD was derived from human ephrin A1, consisting of residues 185 to 205 from the very C terminus, which contains no Lys residues that could react with BS³. Immunoblotting analysis of 293 cells transiently transfected with the ECD.GPI expression vector showed that MAb 12CA5 specifically recognized a band of ~85 kDa (Fig. 7B, compare lane 3 to lane 1). The expected size of fully glycosylated ECD.GPI is ~56 kDa (~40 kDa from glycosylation). Based on the reasoning used for the Cyto protein, we believe that the ~85-kDa protein represents fully glycosylated ECD.GPI protein. After cross-linking, a new band of ~150 kDa was detected (Fig. 7B, lane 4). Since the GPI moiety itself would not be expected to dimerize, we deduce that ECD.GPI dimerizes via the ECD. Nevertheless, to exclude the possibility that the dimerization of ECD.GPI is due to the GPI moiety, the potential of ephrin A1 itself to homodimerize was determined. Immunoblotting analysis of 293 cells transiently transfected with the ephrin A1 expression vector showed that ephrin A1 antibodies specifically recognized a band of ~20 kDa, corresponding to the full-length ephrin A1 protein (Fig. 7B, compare lanes 6 and 5). After cross-linking, a whole array of new bands ranging from ~30 kDa to more than 200 kDa was detected (Fig. 7B, lane 7), probably representing various ephrin A1 hetero-oligomers. In contrast to RPTP ECD.GPI, there was no apparent ephrin A1 homodimer band based on its expected molecular size. Therefore, the homodimerization of the ECD.GPI fusion protein is unlikely to be mediated by the GPI moiety per se, and we conclude that the ECD has an intrinsic ability to homodimerize. Quantitative analysis showed that, when the ECD.GPI fusion protein was expressed on the cell surface to a level similar to that of the FL (Fig. 7C, lanes 1 and 2 versus lanes 3 and 4), FL homodimers but not ECD.GPI homodimers were readily detected after cross-linking (Fig. 7C, lanes 5 and 6 versus lanes 7 and 8), suggesting that ECD by itself has a much weaker dimerization potential than the full-length FL protein.

View larger version (29K): [in this window] [in a new window]   FIG. 7.   The ECD possesses relatively weak dimerization potential and is not required for the homodimerization of the full-length RPTP. (A) A schematic of RPTP constructs used in this figure. (B) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD.GPI (lanes 1 to 4) or ephrin A1 (lanes 5 to 7). Shown are the results of immunoblotting analysis with MAb 12CA5 of whole-cell lysates using ECL detection. (C) In the left panel, transiently transfected 293 cells were biotinylated. Streptavidin-agarose beads were used to isolate the total biotinylated surface proteins, which were then subjected to immunoblotting analysis using MAb 12CA5 followed by 125I-labeled sheep anti-mouse IgG F(ab')2 to determine the levels of surface-expressed RPTP protein. In the right panel, transiently transfected 293 cells were cross-linked with BS3. Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125I-sheep anti-mouse IgG F(ab')2 to determine the levels of RPTP dimers. n.s., nonspecific band. (D) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD. Whole-cell lysates were subjected to immunoblotting analysis with anti-RPTP serum 5478 using ECL detection.

The RPTP TMD efficiently dimerizes in vivo. Some TMDs are known to be able to dimerize (e.g., glycophorin A). To determine whether the TMD also plays a role in RPTP homodimerization, we prepared an expression vector that expresses the RPTP TMD (residues 140 to 164) fused to SN (TMD.SN) (Fig. 8A). Mature SN, a 150 residue bacterial protein, is known to exist exclusively as a monomer and has been fused to other proteins, including the glycophorin A transmembrane domain, for dimerization studies (28, 48). Two stretches (residues 18 to 28 and 131 to 139) from the RPTP ECD were also included in the construct. Residues 18 to 28, immediately C terminal to the signal peptide, are required for correct cleavage of the signal peptide. Residues 131 to 139, containing both polar and charged residues in the juxtamembrane region, are required for membrane localization of the fusion protein. We reasoned that these two short stretches should not obscure our analysis of the TMD, since each of the stretches by themselves is probably too short to form a functional dimerization domain. Moreover, the two stretches are unlikely to interact with each other to form a composite functional dimerization domain, since they are derived from the two extreme ends of the ECD. As for ECD (Fig. 7A), only the  NH₂ group at the N terminus of the mature TMD.SN fusion is left to react with BS³.

View larger version (37K): [in this window] [in a new window]   FIG. 8.   The TMD of RPTP is a potent dimerization domain. (A) A schematic of RPTP constructs used in this figure. (B) 293 cells transiently transfected with TMD.SN were left untreated (lane 1) or were treated with tunicamycin at 200 ng/ml (lane 2) or 1,000 ng/ml (lane 3). Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. Open arrow, most likely a N-glycosylated TMD.SN protein; closed arrow, most likely a nonglycosylated TMD.SN protein; n.s., a nonspecifically recognized band. (C) Mock cross-linking and cross-linking on intact cells by BS3 was performed on 293 cells transiently transfected with pSG5 vector alone or the TMD.SN construct. Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Several studies have strongly suggested that dimerization inhibits the biological activity of certain RPTPs (11, 20, 31, 58). In this study, we showed that homodimers of RPTP can readily be observed after BS³-mediated cross-linking on the surface of intact transiently transfected HEK293 cells. Several lines of evidence suggest that the cross-linking is due to an intrinsic propensity of RPTP to homodimerize: first, the cross-linking of RPTP did not result in heterodimerization with other proteins and was unaffected by the presence of poly-L-lysine, a polymer with many reactive primary NH₂ groups, suggesting that RPTP cross-linking does not occur in a promiscuous fashion (Fig. 1D); second, wedge mutations reduced the cross-linking of RPTP in a fashion stereochemically consistent with our previous crystallographic data (Fig. 3), demonstrating that the cross-linking has strict structural requirements; third, deletion of D2 reduces RPTP homodimerization (Fig. 4), which is consistent with our observation that D2 forms a dimer in crystal structure (A. M. Bilwes, J. den Hertog, T. Hunter, and J. P. Noel, unpublished data); and, finally, in contrast to the cross-linking of ephrin A1-overexpressing cells which resulted in the formation of a whole array of ephrin A1 oligomers (Fig. 7B), cross-linking of cells expressing the many different RPTP constructs consistently resulted in the exclusive formation of homodimers. Furthermore, by performing cross-linking and biotinylation experiments in parallel and by comparing the cross-linking efficiency between wild-type RPTP (FL) and disulfide-bond stabilized RPTP homodimer (FL.137C), we conclude that the majority of cell surface RPTP is homodimerized in transiently transfected 293 cells. In conjunction with previous observations that CD45 also dimerizes (13, 53), these results support the notion that dimerization-mediated negative regulation of PTP activity is physiologically relevant for RPTP as well as for other RPTPs.

We demonstrated that RPTP homodimerization can be mediated by multiple domains, including the ECD, the TMD, D2, and the wedge structure immediately N-terminal to D1. The finding that wedge mutations reduced cross-linking confirms the previous crystallographic data on RPTP (3) as well as functional studies on RPTP (20) and CD45 (31). The finding that deletion of D2 significantly reduced RPTP homodimerization is also consistent with the existence of a D2 crystal dimer. The current study is the first to show that the ECD and the TMD of an RPTP also dimerize. The ECDs of many of the RPTPs are large and contain well-characterized structural moieties such as immunoglobulin-like domains. The ECD of RPTP, however, is short and lacks any obvious structural motifs. It is therefore somewhat unexpected and intriguing that the RPTP ECD dimerizes. The finding that RPTP TMD homodimerizes is reminiscent of the previous reports on some other transmembrane proteins. Dimerization of glycophorin A via its TMD, for example, has been extensively investigated. In fact, many of the dimerization determinants within glycophorin A TMD were mapped using SN fusion proteins, a strategy we adopted in the current study (12, 28, 35). Dimerization via the TMD has also been implicated in the activation of the ErbB2/Neu receptor PTK (60), and FGFR3 (44, 47).

The fact that wedge mutants and D2 deletion mutant had much-reduced dimerization efficiency compared to the full-length RPTP demonstrates that both the wedge and D2 are important although not essential for RPTP homodimerization. However, although we showed that both the ECD and the TMD by themselves can homodimerize, we have been unable to assess the role of these two domains in the context of the native receptor by chemical cross-linking. We cannot compare the cross-linking efficiency of the ECD deletion construct (ECD) and the TMD fusion construct (TMD.SN) to that of the wild-type RPTP (FL) due to the difference in the number of extracellular lysine residues they have available for cross-linking and biotinylation. We have attempted to assess the role of the TMD by inserting single alanine residues into several positions in the TMD of both the full-length RPTP and TMD.SN fusion construct, but none of these single insertions reduced the level of cross-linked homodimers significantly. Other experimental approaches, such as fluorescence resonance energy transfer (FRET), will be needed to study RPTP proteins with a modified ECD and TMD. In this connection, using RPTP chimeras in which the ECD, TMD, and D1 of RPTP are fused to two different GFP derivatives, we have recently shown that RPTP dimerization can be detected in living cells by FRET analysis, and, by analyzing a panel of deletion mutants, found that the TMD was required and sufficient for dimerization of these chimeras (L. G. Tertoolen, J. C. Blanchetot, G. Jiang, J. Overvoorde, T. W. J. Gadella, T. Hunter, and J. den Hertog, submitted for publication).

So far, we have not determined the exact contribution of each of the individual dimerization domains toward the stable homodimerization of RPTP. One of the difficulties in reaching a clear conclusion lies in our observation that a structural domain in isolation may behave somewhat differently than in the context of the full-length receptor. For instance, truncation mutants lacking the entire cytoplasmic domain (Cyto, Fig. 5) dimerized with high efficiency, suggesting that the cytoplasmic domain is not essential for dimerization. On the other hand, both wedge mutations and the D2 deletion significantly reduced RPTP dimerization potential (Fig. 3 and 4), suggesting otherwise. We believe that it is reasonable to assume that results based on the mutated full-length receptor forms are more relevant than those based on truncated receptors or isolated domains. Accordingly, it appears that the wedge structure and D2 may have a relatively larger contribution than the ECD and/or TMD toward the stability of the dimer of the full-length receptor. Although this appears to be inconsistent with the fact that the Cyto truncation mutant homodimerizes as efficiently as the wild-type protein, the lack of bulky cytoplasmic domains may allow the TMDs to be aligned more closely in the truncated protein dimer than in full-length RPTP, therefore exaggerating the TMD-TMD interaction.

The fact that cross-linking of transiently expressed full-length RPTP was observed in the absence of added ligand(s) suggests that RPTP homodimerizes in a ligand-independent fashion. However, given that several widely expressed surface proteins and extracellular matrix components have been found to bind to and potentially act as ligands for RPTPs (1, 34, 38, 40, 41, 62), one cannot exclude the possibility that homodimerization of full-length RPTP is actually mediated by an unidentified ligand present in the tissue culture system. The fact that the RPTP ECD deletion mutant still dimerized (Fig. 7D) unequivocally demonstrates that the efficient homodimerization of the truncated RPTP can occur in a ligand-independent fashion. However, since some truncated receptor PTKs lacking all or part of the ECD undergo ligand-independent dimerization, whereas the full-length receptors do not (15, 22, 57), the fact that the RPTP ECD deletion mutant dimerized does not necessarily imply that the dimerization of full-length RPTP is also ligand independent. So far, we have been unable to cross-link endogenous RPTP in embryonic fibroblasts or retrovirally transduced RPTP in RPTP^/ embryonic fibroblasts derived from RPTP^/ mice (50). One possible explanation is that cross-linked dimers exist but are below the limit of detection, since we only detected a fairly faint band of the monomeric RPTP protein in these cells. Another reason may be that these cells express a secreted ligand that promotes its dissociation or an intracellular protein that binds RPTP and prevents dimerization that is absent or present at lower levels in 293 cells. Indeed, it may be necessary to overexpress RPTP to override such dissociation factors and thereby detect RPTP dimerization. The reported association of RPTP with contactin via its ECD is an example of an interaction that might prevent RPTP dimerization (62). Likewise, proteins interacting with the intracellular domain of RPTP, in a manner similar to the interaction of LIP1 and the catenin-cadherin complex with intracellular domain of LAR (25, 46), could reduce the extent of RPTP dimerization.

In conjunction with our previous observation that dimerization inhibits RPTP biological activity (20), the current results lead us to propose that, in its inactive state, RPTP exists as homodimers. Furthermore, based on the observation that multiple domains appears to mediate RPTP homodimerization, we propose a zipper model in which RPTP homodimers are stabilized by weak interactions between multiple dimer interfaces (Fig. 9A). In addition to the types of symmetric interactions depicted, it remains to be seen whether asymmetric interactions, such as a D1-D2 interaction, may also play a role in RPTP intermolecular interactions, as suggested by studies of RPTP and RPTP (58). The inactive dimeric state of RPTP could in principle be induced by ligand binding (Fig. 9A, left). If this were true, we note that it is the opposite of how ligands regulate receptor PTK activity, where ligand-induced dimerization leads to activation (16) (Fig. 9B). Alternatively, based on the zipper model where multiple domains are involved in receptor homodimerization, it is possible that RPTP is constitutively dimeric and inactive (Fig. 9A, right top). In this case, RPTP may be activated by ligand(s) that stabilize the monomeric state of the receptor, thus preventing dimerization, or by an intracellular signaling event(s), such as phosphorylation, that induces an open conformation of the intracellular domains (Fig. 9A, right). In fact, we showed that both the EGF-bound EGFR-CD45 chimeric receptor (31) and disulfide-bonded full-length RPTP dimers (20) with wedge mutation(s) are biologically active even though they are dimerized via their ECDs, supporting the notion that RPTP dimers can be enzymatically active. Considering that wedge mutations reduce dimerization efficiency as much as 80% (Fig. 3), it is possible that active dimers are unstable and may act as a transition state between inactive dimers and active monomers. The notion that RPTP dimerization may be regulated by intracellular signaling events is particularly attractive for receptors such as RPTP, which has a short ECD without any recognizable protein-protein interaction domains. In fact, we showed previously that RPTP is activated by phosphorylation of Ser180 and Ser204, which lie in the juxtamembrane domain close to the wedge (9, 55). We are currently testing whether such phosphorylation can decrease RPTP homodimerization.

View larger version (27K): [in this window] [in a new window]   FIG. 9.   A model for the regulation of RPTPs via dimerization. (A) Hypothetical model for regulation of RPTPs via dimerization. In the inactive state, RPTPs are dimerized via wedge-active site interaction in D1, interaction via the TMD, and interaction via the ECD. In the active state, the receptors are either monomers or dimers in which dimerization via D1 no longer occurs due to phosphorylation. Ligand binding can either destabilize or stabilize dimers. (B) Classical model of activation of receptor PTKs. Ligand binding leads to receptor dimerization, transautophosphorylation, and subsequent kinase activation. KD, kinase domain; L, ligand; P, phosphorylation.

In summary, we provide evidence that RPTP has the potential to homodimerize efficiently in vivo via multiple interacting domains, including the wedge structure, the TMD, and the ECD. The results presented here are consistent with our observation that dimerization decreases catalytic activity via a wedge region interaction (20), as we originally proposed (3). The results therefore suggest that dimerization-mediated downregulation of RPTP biological activity is most likely physiologically relevant. Given that dimerization has been shown to inhibit several different RPTPs and that the wedge element is conserved, we believe that there will be other examples of RPTPs that are regulated in a similar manner through dimerization. However, this does not mean that all RPTPs will be regulated in this fashion. For instance, D1 of RPTPµ does not form a wedge-mediated dimer in the crystal structure (17), even though the helix-turn-helix that forms the wedge is present, and instead forms a different type of dimer. Additionally, the cytoplasmic portion (D1+D2) of LAR did not form a dimer in the recently reported crystal structure (37). Thus, it will obviously be important to determine which RPTPs can dimerize and whether this inhibits their activity and to investigate exactly how RPTP dimerization is regulated. In this connection, it will be very interesting to determine whether pleiotrophin, which binds to RPTP and inhibits its activity (33), induces RPTP dimerization.